Sato S I, Nam J W, Kasuga K, Nojiri H, Yamane H, Omori T
Biotechnology Research Center, The University of Tokyo, Bunkyo-ku, Japan.
J Bacteriol. 1997 Aug;179(15):4850-8. doi: 10.1128/jb.179.15.4850-4858.1997.
Nucleotide sequence analysis of the flanking regions of the carBC genes of Pseudomonas sp. strain CA10 revealed that there were two open reading frames (ORFs) ORF4 and ORF5, in the upstream region of carBC. Similarly, three ORFs, ORF6 to ORF8, were found in the downstream region of carBC. The deduced amino acid sequences of ORF6 and ORF8 showed homologies with ferredoxin and ferredoxin reductase components of bacterial multicomponent dioxygenase systems, respectively. ORF4 and ORF5 had the same sequence and were tandemly linked. Their deduced amino acid sequences showed about 30% homology with large (alpha) subunits of other terminal oxygenase components. Functional analysis using resting cells harboring the deleted plasmids revealed that the products of ORF4 and -5, ORF6, and ORF8 were terminal dioxygenase, ferredoxin, and ferredoxin reductase, respectively, of carbazole 1,9a-dioxygenase (CARDO), which attacks the angular position adjacent to the nitrogen atom of carbazole, and that the product of ORF7 is not indispensable for CARDO activity. Based on the results, ORF4, ORF5, ORF6, and ORF8 were designated carAa, carAa, carAc, and carAd, respectively. The products of carAa, carAd, and ORF7 were shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be polypeptides with molecular masses of 43, 36, and 11 kDa, respectively. However, the product of carAc was not detected in Escherichia coli. CARDO has the ability to oxidize a wide variety of polyaromatic compounds, including dibenzo-p-dioxin, dibenzofuran, biphenyl, and polycyclic aromatic hydrocarbons such as naphthalene and phenanthrene. Since 2,2',3-trihydroxydiphenyl ether and 2,2',3-trihydroxybiphenyl were identified as metabolites of dibenzo-p-dioxin and dibenzofuran, respectively, it was considered that CARDO attacked at the angular position adjacent to the oxygen atom of dibenzo-p-dioxin and dibenzofuran as in the case with carbazole.
对假单胞菌属菌株CA10的carBC基因侧翼区域进行核苷酸序列分析发现，在carBC的上游区域存在两个开放阅读框（ORF），即ORF4和ORF5。同样，在carBC的下游区域发现了三个ORF，即ORF6至ORF8。ORF6和ORF8推导的氨基酸序列分别与细菌多组分双加氧酶系统的铁氧还蛋白和铁氧还蛋白还原酶组分具有同源性。ORF4和ORF5具有相同的序列且串联相连。它们推导的氨基酸序列与其他末端加氧酶组分的大亚基（α亚基）显示出约30%的同源性。使用携带缺失质粒的静息细胞进行功能分析表明，ORF4和-5、ORF6以及ORF8的产物分别是咔唑1,9a-双加氧酶（CARDO）的末端双加氧酶、铁氧还蛋白和铁氧还蛋白还原酶，CARDO作用于咔唑氮原子相邻的角位，并且ORF7的产物对于CARDO活性并非不可或缺。基于这些结果，ORF4、ORF5、ORF6和ORF8分别被命名为carAa、carAa、carAc和carAd。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示，carAa、carAd和ORF7的产物分别是分子量为43 kDa、36 kDa和11 kDa的多肽。然而，在大肠杆菌中未检测到carAc的产物。CARDO能够氧化多种多环芳烃化合物，包括二苯并-对-二恶英、二苯并呋喃、联苯以及萘和菲等多环芳烃。由于分别鉴定出2,2',3-三羟基二苯醚和2,2',3-三羟基联苯是二苯并-对-二恶英和二苯并呋喃的代谢产物，因此认为CARDO与咔唑的情况一样，作用于二苯并-对-二恶英和二苯并呋喃氧原子相邻的角位。
